Methods of treating influenza

ABSTRACT

The present invention relates to the treatment or prevention of influenza virus infection. In particular, the present invention relates to the use of N-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or a pharmaceutically acceptable salt thereof, in the treatment or prevention of influenza virus infection.

FIELD OF THE INVENTION Cross Reference to Related Applications

This is the § 371 U.S. National Stage of International Application No.PCT/AU2018/050085, filed Feb. 7, 2018, which was published in Englishunder PCT Article 21(2), which in turn claims priority from AustralianProvisional Patent Application No. 2017900385, filed Feb. 8, 2017, thecontents of which are incorporated in their entirety herein.

The present invention relates to the treatment or prevention ofinfluenza virus infection. In particular, the present invention relatesto antiviral compounds and their use in the treatment or prevention ofinfluenza virus infection.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should inno way be considered as an admission that such prior art is widely knownor forms part of common general knowledge in the field.

Influenza is one of the few common infectious diseases poorly controlledby modern medicine. The burden of Influenza is heavy, with the WorldHealth Organisation estimating 5-10% of adults and 20-30% of children inthe global population (3-5 million people) are affected by annualepidemics, causing between 250,000-500,000 deaths per year.

Symptoms of influenza infection can be mild to severe. The most commonsymptoms include: a high fever, runny nose, sore throat, muscle pains,headache, coughing, and fatigue. Complications associated with fluinclude pneumonia, bronchitis, sinus infections and ear infections.Other possible serious complications triggered by flu can includeinflammation of the heart, brain or muscle, and multi-organ failure.Infection of the respiratory tract can trigger an extreme inflammatoryresponse in the body and can lead to sepsis. Flu can also make chronicmedical problems worse. For example, people with asthma may experienceasthma attacks while they have the flu, and people with chronic heartdisease may experience a worsening of this condition triggered by flu.

Influenza is caused by viruses of the family Orthomyxoviridae, and thereare three types of influenza viruses that affect people, called Type A,Type B, and Type C. These influenza viruses generally lead to similarsymptoms but the virus types are unrelated antigenically, so thatinfection with one type confers no immunity against the other. InfluenzaA and B viruses cause seasonal epidemics almost every winter. Theemergence of a new and very different influenza A virus to infect peoplecan cause an influenza pandemic. Influenza type C infections generallycause a mild respiratory illness and are not thought to cause epidemics.

Influenza A viruses can be broken down into different subtypes accordingto genetic variations of two different viral surface proteins:hemagglutinin (HA/H in subtype) and neuraminidase (NA/N in subtype).There are 16 different HAs and 9 NAs which are distinguishableserologically. Influenza B viruses are not divided into subtypes, butcan be broken down into lineages. Currently circulating influenza Bviruses belong to one of two lineages: B/Yamagata and B/Victoria.Further variation exists and specific influenza strain isolates areidentified by a standard nomenclature specifying virus type, host oforigin, geographical origin, sequential number of isolation, year ofisolation, and for influenza A the HA and NA subtype.

When a new strain of influenza virus appears, human populations havelittle native resistance and existing influenza vaccines often havelimited efficacy as they are typically active against specific influenzaA and B viruses that differ from those in the emerging strain. Thisphenomenon has led to epidemics that occur regularly. Moreover,influenza viruses undergo a gradual antigenic variation (antigenicdrift) that degrades the level of immunological resistance againstrenewed infection. Pandemics occur every couple of decades and are dueto a dramatic change (antigenic shift) in the viral HA and NA subtypes.

Anti-influenza vaccines, containing killed strains of types A and Bvirus currently in circulation, are available, but have only a 50 to 60%success rate in preventing infection. Standard influenza vaccines haveto be redesigned each year to counter new variants of the virus. Inaddition, any immunity provided is short-lived. Therefore, vaccines maynot prevent or limit a pandemic if the circulating strains continue todrift significantly, or another subtype emerges. Instead, antiviralagents will be critical for initial control and protection against anemerging pandemic.

Two classes of antivirals are approved for clinical use against humaninfluenza:

Neuraminidase Inhibitors—including oseltamivir, zanamivir and peramivir.

M2 Ion-Channel Inhibitors—including amantadine and rimantadine. Thesemedications are active against influenza A viruses, but not influenza Bviruses due to structurally distinct M2 channels.

Oseltamivir has become the preferred choice of neuraminidase inhibitorfor governments and organizations in their preparations for possibleinfluenza pandemics due to its oral delivery. Stockpiles of oseltamivirwere utilised during the 2009 influenza A H1N1 pandemic (Nguyen-Van-Tamet al., Clin Microbiol Infect, 2015. 21: 222-22). However, the clinicaleffectiveness of neuraminidase inhibitors, such as oseltamivir, is stillundergoing debate. Recent reports have shown that oseltamivir reducesthe time to alleviation of symptoms of influenza in adults by only 16.8hours (Jefferson et al., Cochrane Database Syst Rev 2014; 4) and did notsignificantly reduce incidence of pneumonia, serious complications,hospitalizations or deaths (Nguyen-Van-Tam et al., Clin MicrobiolInfect, 2015. 21: 222-225). Further, the use of oseltamivir has beenshown to increase the risk of adverse effects, such as nausea, vomiting,psychiatric effects and renal events in adults and vomiting in children(Jefferson et al., Cochrane Database Syst Rev 2014; 4).

Prior to 2007, resistance to neuraminidase inhibitors among influenzaviruses was generally low, ˜1% (Ilyushina et al., Antivir Res, 2006.70(3): 121-131; Stiver, CMAJ, 2003. 168(1):49-57). However, the Centresfor Disease Control and Prevention reported in 2007-2008 that influenzaA (H1N1) showed an emergence and worldwide spread of oseltamivirresistance. Oseltamivir-resistant influenza A viruses carrying the H275YNA mutation emerged as a result of genetic drift and drug treatment.Since the emergence of this oseltamivir-resistant influenza virus strainthere is concern that the H275Y substitution may generate a futurepandemic or even become fixed in the viral genome. This brings intoquestion the rationale for stockpiling neuraminidase inhibitors(Nguyen-Van-Tam et al., Clin Microbiol Infect, 2015. 21: 222-225), andhighlights the need to continuously monitor antiviral drugsusceptibilities and prioritise research for novel influenza antiviraltherapeutics (Samson, et al. Antiviral Res. 2013, 98(2):174-85).

Unlike neuraminidase inhibitors, amantadine and rimantadine areinexpensive and widely available. Amantadine is a derivative ofadamantane that efficiently inhibits replication of influenza A viruses(Davies et al., Science, 1964. 144: 862-863). The drug inhibitsreplication by directly binding to the influenza virus M2 protein in theenvelope membrane and blocking its H⁺-conductive ion channel activitythat is essential for virus replication (Sugrue & Hay, Virology, 1991.180: 617-624; Pinto et al., Cell, 1992. 69: 517-528; Schroeder et al., JGen Virol, 1994. 75: 3477-3484). At higher concentrations amantadinealso indirectly increases endosomal pH, which alters the pH-sensitiveconformation of hemagglutinin reducing the release of infectious virusparticles (Daniels et al., Cell, 1985. 40: 431-439; Steinhauer et al.,Proc Natl Acad Sci, 1991. 88: 11525-11529).

However, amantadine and rimantadine are only effective against influenzaA and drug-resistant variants can be obtained easily by culturing thevirus in the presence of these agents (Cochran et al., Ann NY Acad Sci,1965. 130(1): 432-439; Appleyard, J Gen Virol, 1977. 36: 249-255).Amantadine and rimantadine resistance develops as a result of mutationsin the transmembrane region of M2 that preserve the ion channel activitywhile reducing the ability of the drugs to inhibit virus replication.The frequency of amantadine-resistant influenza A virus strains foundcirculating in the human population rose from 0.4% in 1995 to 12.3% in2003-2004 (Bright et al., Lancet. 2005. 366, 1175-1181). In isolatesfrom Asia, 61% of strains possessed mutations that conferred amantadineresistance.

There is a need for new antiviral therapies that are effective in thetreatment of multiple subtypes and strains of influenza, in particularsubtypes and strains of influenza that are resistant to currenttherapies.

It is an object of the present invention to overcome or ameliorate atleast one of the disadvantages of the prior art, or to provide a usefulalternative.

SUMMARY OF THE INVENTION

Previously, N-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide(BIT225) has been shown to have only limited activity against the M2viroporin of influenza A and was not considered to be suitable fortreating influenza (Jalily et al., MolPharm, 2016. 90: 80-95). However,the present application surprisingly shows that BIT225 has activityagainst multiple subtypes and strains of influenza virus. Importantly,BIT225 has been found to have activity against subtypes and strains thatare resistant to amantadine.

The chemical structure of BIT225 is shown below:

BIT225 may also be referred to asN-carbamimidoyl-5-(1-methylpyrazol-4-yl)naphthalene-2-carboxamide or5-(1-methylpyrazol-4-yl)2-naphthoylguanidine.

The present invention generally relates to the use of BIT225, or apharmaceutically acceptable salt thereof, for treating or preventinginfluenza, for inhibiting the replication of influenza virus, forreducing the severity, intensity, or duration of complications orsymptoms associated with influenza, or for reducing the titre ofinfluenza virus.

According to one aspect, the present invention provides a method for thetreatment or prevention of influenza virus infection in a subject, themethod comprising administering to the subject an effective amount ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof.

According to another aspect, the present invention provides a method forinhibiting the replication of influenza virus in a subject, the methodcomprising administering to the subject an effective amount ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof.

According to another aspect, the present invention provides a method forreducing the severity, intensity, or duration of complications orsymptoms associated with influenza virus infection in a subject, themethod comprising administering to the subject an effective amount ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof.

According to another aspect, the present invention provides a method ofreducing the titre of influenza virus in a subject, the methodcomprising administering to the subject an effective amount ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof.

According to another aspect, the present invention provides use ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment or prevention of influenza virus infection.

According to another aspect, the present invention provides use ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for inhibiting the replication of influenza virus.

According to another aspect, the present invention provides use ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for reducing the severity, intensity, or duration ofcomplications or symptoms associated with influenza virus infection.

According to another aspect, the present invention provides use ofN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for reducing the titre of influenza virus.

According to another aspect, the present invention provides acomposition comprisingN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for use in a method oftreating or preventing influenza virus infection in a subject.

According to another aspect, the present invention provides acomposition comprisingN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for use in a method ofinhibiting the replication of influenza virus in a subject.

According to another aspect, the present invention provides acomposition comprisingN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for use in a method ofreducing the severity, intensity, or duration of complications orsymptoms associated with influenza virus infection in a subject.

According to another aspect, the present invention provides acomposition comprisingN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, for use in a method ofreducing the titre of influenza virus in a subject.

In certain embodiments, the influenza virus is selected from influenza Avirus, influenza B virus and influenza C virus.

In certain embodiments, the influenza virus is influenza A virus of anyhost origin (human, swine, chicken, equine etc).

In certain embodiments, the influenza A virus is selected from H1N1,H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H7N1, H7N2,H7N3, H7N4, H7N7, H7N9, H9N2, and H10N7 subtypes.

In certain embodiments, the influenza A virus is selected from H1N1,H3N2 and H5N1 subtypes.

In certain embodiments, the influenza virus is influenza B virus of anylineage.

In certain embodiments, the influenza B virus is selected fromB/Yamagata and B/Victoria lineages.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered by a routeselected from oral, nasal, intravenous, intraperitoneal, inhalation andtopical.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered daily.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered twice daily.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered at a dosage ofabout 100 mg to about 600 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally and isadministered at a dosage of about 600 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally and isadministered at a dosage of about 200 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally and isadministered at a dosage of about 100 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally and isadministered daily.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally and isadministered twice daily.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally, oncedaily at a dosage of about 200 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally, twicedaily at a dosage of about 200 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally, oncedaily at a dosage of about 100 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered orally, twicedaily at a dosage of about 100 mg.

In certain embodiments, theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, is administered in combinationwith one or more additional antiviral agents.

In certain embodiments, the additional antiviral agents are selectedfrom zanamivir, oseltamivir and peramivir.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: This figure demonstrates the EC₅₀ for BIT225, which wasapproximately 10 μM against both the wild-type and A30T mutant viruses,with respective EC₉₀ values of 12 μM and 18 μM. This is in starkcontrast to the large shift in drug sensitivity to amantadine caused bythe A30T mutation in M2.

FIG. 2: This figure shows inhibition by BIT225 of replication in a Tcell line (C8166) of the chimeric viruses SHIV-1_(M2) andSHIV-1_(SCVpu), which express the ion channel sequences of influenza AM2 or HIV-1 Vpu, respectively. Treatment with 10 μM BIT225 reduced virusreleased into the culture supernatant as measured by viral p27 protein.Controls treated with carrier solvent alone (DMSO) are shown forcomparison

FIG. 3: This figure shows that treatment with 10 μM BIT225 of the mutantSHIV-1_(PND2) virus with a non-functional Vpu ion channel does notsignificantly reduce virus replication.

DEFINITIONS

In describing and claiming the present invention, the followingterminology has been used in accordance with the definitions set outbelow. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly and is not intended to be limiting. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one having ordinary skill in the art to which theinvention pertains.

In the context of the present invention, the words “comprise”,“comprising” and the like are to be construed in their inclusive, asopposed to their exclusive, sense, that is in the sense of “including,but not limited to”.

The terms “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The term “influenza virus” as used herein, refers to an RNA virus of theOrthomyxoviridae family, including influenza A, influenza B, andinfluenza C, and mutants thereof. Influenza A viruses contemplatedherein include those viruses that have two antigenic glycosylatedenzymes on their surface: neuraminidase and hemagglutinin. Varioussubtypes of influenza A virus that can be treated using the materialsand methods of the subject invention include, but are not limited to,H1N1, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H7N1,H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, and H10N7. Influenza B virusesinclude, but are not limited to, B/Yamagata and B/Victoria lineages.

As used herein, the term “influenza” or “flu” refers to an infectiousdisease caused by an influenza virus.

The term “symptom(s)” as used herein, refers to signs or indicationsthat a subject is suffering from a specific condition or disease. Forexample, symptoms associated with an influenza infection, as usedherein, refer to signs or indications that a subject is infected with aninfluenza virus. Influenza-related symptoms contemplated herein include,but are not limited to, fever, headache, exhaustion/fatigue, muscularaches, sore joints, irritated watering eyes, malaise, nausea and/orvomiting, shaking, chills, chest pain, sneezing and respiratory symptoms(i.e., inflamed respiratory mucous membranes, substernal burning, nasaldischarge, scratchy/sore throat, dry cough, loss of smell).

Symptoms associated with an influenza infection can start within 24 to48 hours after infection and can begin suddenly. Chills or a chillysensation are often the first indication of influenza. Fever is commonduring the first few days, and the temperature may rise to 39° C. Inmany instances, subjects feel sufficiently ill to remain in bed fordays; subjects often experience aches and pains throughout the body,most pronounced in the back and legs.

As used herein, the term “complication(s)” refers to a pathologicalprocess or event occurring during a disease or condition that is not anessential part of the disease or condition; where it may result from thedisease/condition or from independent causes. Accordingly, the termcomplication(s) refers to medical/clinical problems that are observed insubjects diagnosed with an influenza infection. One complication of aninfluenza infection is that the influenza infection can make chronichealth problems worse. For example, complications associated with aninfluenza infection include, without limitation, encephalitis,bronchitis, tracheitis, myositis rhinitis, sinusitis, asthma, bacterialinfections (i.e., streptococcus aureus bacterial infection, Haemophilusinfluenzae bacterial infection, staphylococcal pneumonia bacterialinfection), cardiac complications (i.e., atrial fibrillation,myocarditis, pericarditis), Reye's syndrome, neurologic complications(i.e., confusion, convulsions, psychosis, neuritis, Guillain-Barresyndrome, coma, transverse myelitis, encephalitis, encephalomyelitis),toxic shock syndrome, myositis, myoglobinuria, and renal failure, croup,otitis media, viral infections (i.e., viral pneumonia), pulmonaryfibrosis, obliterative bronchiolitis, bronchiectasis, exacerbations ofasthma, exacerbations of chronic obstructive pulmonary disease, lungabscess, empyema, pulmonary aspergillosis, myositis and myoglobinaemia,heart failure, early and late fetal deaths in pregnant women, increasedperinatal mortality in pregnant women, and congenital abnormalities inbirth.

As used herein, the term “effective amount” in the context ofadministering a therapy to a subject refers to the amount of a therapywhich has a prophylactic and/or therapeutic effect(s). In certainembodiments, an “effective amount” in the context of administration of atherapy to a subject refers to the amount of a therapy which issufficient to achieve one, two, three, four, or more of the followingeffects: (i) reduce or ameliorate the severity of an influenza virusinfection, disease or symptom associated therewith; (ii) reduce theduration of an influenza virus infection, disease or symptom associatedtherewith; (iii) prevent the progression of an influenza virusinfection, disease or symptom associated therewith; (iv) causeregression of an influenza virus infection, disease or symptomassociated therewith; (v) prevent the development or onset of aninfluenza virus infection, disease or symptom associated therewith; (vi)prevent the recurrence of an influenza virus infection, disease orsymptom associated therewith; (vii) reduce or prevent the spread of aninfluenza virus from one cell to another cell, one tissue to anothertissue, or one organ to another organ; (ix) prevent or reduce the spreadof an influenza virus from one subject to another subject; (x) reduceorgan failure associated with an influenza virus infection; (xi) reducehospitalization of a subject; (xii) reduce hospitalization length;(xiii) increase the survival of a subject with an influenza virusinfection or disease associated therewith; (xiv) eliminate an influenzavirus infection or disease associated therewith; (xv) inhibit or reduceinfluenza virus replication; (xvi) inhibit or reduce the entry of aninfluenza virus into a host cell(s); (xviii) inhibit or reducereplication of the influenza virus genome; (xix) inhibit or reducesynthesis of influenza virus proteins; (xx) inhibit or reduce assemblyof influenza virus particles; (xxi) inhibit or reduce release ofinfluenza virus particles from a host cell(s); (xxii) reduce influenzavirus titre; and/or (xxiii) enhance or improve the prophylactic ortherapeutic effect(s) of another therapy.

In certain embodiments, the effective amount does not result in completeprotection from an influenza virus disease, but results in a lower titreor reduced number of influenza viruses compared to an untreated subject.In certain embodiments, the effective amount results in a 0.5 fold, 1fold, 2 fold, 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greaterreduction in titre of influenza virus relative to an untreated subject.In some embodiments, the effective amount results in a reduction intitre/volume or weight of sample of influenza virus relative to anuntreated subject of approximately 1 log or more, approximately 2 logsor more, approximately 3 logs or more, approximately 4 logs or more,approximately 5 logs or more, approximately 6 logs or more,approximately 7 logs or more, approximately 8 logs or more,approximately 9 logs or more, approximately 10 logs or more, 1 to 3logs, 1 to 5 logs, 1 to 8 logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs,2 to 7 logs, 2 logs to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3to 7 logs, 3 to 8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9logs, 5 to 6 logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6to 8 logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.Benefits of a reduction in the titre, number or total burden ofinfluenza virus include, but are not limited to, less severe symptoms ofthe infection, fewer symptoms of the infection and a reduction in thelength of the disease associated with the infection.

“Concurrent administration”, “concurrently administering”,“co-administration”, “co-administered” and the like as used herein,includes administering BIT225, or a pharmaceutically acceptable saltthereof, and one or more additional viral therapeutics together in amanner suitable for the treatment of an influenza infection or for thetreatment of influenza infection-related symptoms/complications. Ascontemplated herein, concurrent administration includes providing to asubject BIT225, or a pharmaceutically acceptable salt thereof, and oneor more additional viral therapeutics as separate compounds, such as,for example, separate pharmaceutical compositions administeredconsecutively, simultaneously, or at different times. Preferably, ifBIT225, or a pharmaceutically acceptable salt thereof, and one or moreadditional viral therapeutic are administered separately, they are notadministered so distant in time from each other that BIT225, or apharmaceutically acceptable salt thereof, and the one or more additionalviral therapeutic cannot interact. BIT225, or a pharmaceuticallyacceptable salt thereof, and one or more additional viral therapeuticmay be administered in any order. In one embodiment, BIT225, or apharmaceutically acceptable salt thereof, can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after)the administration the one or more additional viral therapeutics to asubject. According to the subject invention, concurrent administrationalso encompasses providing one or more additional viral therapeutics inadmixture with BIT225, or a pharmaceutically acceptable salt thereof,such as in a pharmaceutical composition.

An additional viral therapeutic of the invention includes vaccinationsor antiviral medications such as a neuraminidase or hemagglutinininhibitor or medications that modulate the immune system or host cellfactors. Contemplated viral therapeutics for use in accordance with thesubject invention include, but are not limited to, amantadine,rimantadine, ribavirin, idoxuridine, trifluridine, vidarabine,acyclovir, ganciclovir, foscarnet, zidovudine, didanosine, peramivir,zalcitabine, stavudine, famciclovir, oseltamivir, zanamivir, andvalaciclovir.

In related embodiments, a subject diagnosed with an influenza infection,BIT225, or a pharmaceutically acceptable salt thereof, may beconcurrently administered with other therapeutics useful in thetreatment of symptoms associated with an influenza infection. Forexample, antitussives, mucolytics, expectorants, antipyretics,analgesics, or nasal decongestants can be concurrently administered withBIT225, or a pharmaceutically acceptable salt thereof, to a subjectdiagnosed with an influenza infection.

As used herein, the term “infection” means the invasion by,multiplication and/or presence of a virus in a cell or a subject. In oneembodiment, an infection is an “active” infection, i.e., one in whichthe virus is replicating in a cell or a subject. Such an infection ischaracterized by the spread of the virus to other cells, tissues, and/ororgans, from the cells, tissues, and/or organs initially infected by thevirus. An infection may also be a latent infection, i.e., one in whichthe virus is dormant.

As used herein, the expression “treating influenza virus infection”means improving, reducing, or alleviating at least one symptom orbiological consequence of influenza virus infection in a subject, and/orreducing or decreasing influenza virus titer, load, replication orproliferation in a subject following exposure to an influenza virus. Theexpression “treating influenza virus infection” also includes shorteningthe time period during which a subject exhibits at least one symptom orbiological consequence of influenza virus infection after being infectedby influenza virus. Methods for treating influenza virus infection,according to the present invention, comprise administering apharmaceutical composition of the present invention to a subject afterthe subject is infected with an influenza virus and/or after the subjectexhibits or is diagnosed with one or more symptoms or biologicalconsequences of influenza virus infection.

As used herein, the expression “preventing influenza virus infection”means preventing at least one symptom or biological consequence ofinfluenza virus infection in a subject, and/or inhibiting or attenuatingthe extent to which influenza virus is capable of entering, spreading,and/or propagating within/among cells of an animal body. The expression“preventing influenza virus infection” also includes decreasing thesusceptibility of a subject to at least one symptom or biologicalconsequence of influenza virus infection. Methods for preventinginfluenza virus infection (i.e., prophylaxis) comprise administering apharmaceutical composition of the present invention to a subject beforethe subject is infected with an influenza virus and/or before thesubject exhibits one or more symptoms or biological consequences ofinfluenza virus infection. Methods for preventing influenza virusinfection may include administering a pharmaceutical composition of thepresent invention to a subject at a particular time period or season ofthe year (e.g., during the 1-2 month period just prior to the time atwhich peak numbers of individuals are typically found to experienceinfluenza virus infection), or before the subject travels to or isexposed to an environment with high frequencies of influenza virusinfection, and/or before the subject is exposed to other subjects whoare infected with influenza virus.

As used herein, the terms “replication,” “viral replication” and “virusreplication” in the context of a virus refer to one or more, or all, ofthe stages of a viral life cycle which result in the propagation ofvirus. The steps of a viral life cycle include, but are not limited to,virus attachment to the host cell surface, penetration or entry of thehost cell (e.g., through receptor mediated endocytosis or membranefusion), uncoating (the process whereby the viral capsid is removed anddegraded by viral enzymes or host enzymes thus releasing the viralgenomic nucleic acid), genome replication, synthesis of viral messengerRNA (mRNA), viral protein synthesis, and assembly of viralribonucleoprotein complexes for genome replication, assembly of virusparticles, post-translational modification of the viral proteins, andrelease from the host cell by lysis or budding and acquisition of aphospholipid envelope which contains embedded viral glycoproteins. Insome embodiments, the terms “replication,” “viral replication” and“virus replication” refer to the replication of the viral genome. Inother embodiments, the terms “replication,” “viral replication” and“virus replication” refer to the synthesis of viral proteins.

As used herein, the term “titre” in the context of a virus refers to thenumber of viral particles present in a given volume of blood or otherbiological fluid or tissue or organ weight. The terms “viral load” and“viral burden” may also be used.

As used herein, the term “subject” is used to refer to an animal (e.g.,birds, reptiles, and mammals). In a specific embodiment, a subject is abird. In another embodiment, a subject is a mammal including anon-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep,cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, anda human). In certain embodiments, a subject is a non-human animal. Insome embodiments, a subject is a farm animal or pet. In anotherembodiment, a subject is a human. In another embodiment, a subject is ahuman infant. In another embodiment, a subject is a human child. Inanother embodiment, a subject is a human adult. In another embodiment, asubject is an elderly human. In another embodiment, a subject is apremature human infant.

Routes of administration include, but are not limited to, intravenous,intraperitoneal, subcutaneous, intracranial, intradermal, intramuscular,intraocular, intrathecal, intracerebral, intranasal, transmucosal, or byinfusion orally, rectally, via iv drip, patch and implant. Oral routesare particularly preferred.

Compositions suitable for injectable use include sterile aqueoussolutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol and liquidpolyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed by, forexample, filter sterilization or sterilization by other appropriatemeans. Dispersions are also contemplated and these may be prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, a preferredmethod of preparation includes vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution.

When the active ingredients are suitably protected, they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets. For oral therapeuticadministration, the active compound may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 0.01% by weight,more preferably 0.1% by weight, even more preferably 1% by weight ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 1 toabout 99%, more preferably about 2 to about 90%, even more preferablyabout 5 to about 80% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between about 0.1 ng and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

The present invention also extends to forms suitable for topicalapplication such as creams, lotions and gels. In such forms, componentsmay be added or modified to assist in penetration of the surfacebarrier.

Procedures for the preparation of dosage unit forms and topicalpreparations are readily available to those skilled in the art fromtexts such as Pharmaceutical Handbook, 19^(th) edition (Edited by AinleyWade), The Pharmaceutical Press London; CRC Handbook of Chemistry andPhysics (edited by Robert C. Weast), CRC Press Inc.; Goodman andGilman's The Pharmacological basis of Therapeutics, 9^(th) edition,McGraw Hill; Remington: The Science and Practice of Pharmacy, 19^(th)edition (edited by Joseph P. Remington and Alfonso R. Gennaro), MackPublishing Co.

The term “pharmaceutically acceptable salt,” as used herein, refers toany salt of BIT225 that is pharmaceutically acceptable and does notgreatly reduce or inhibit the activity BIT225. Suitable examples includeacid addition salts, with an organic or inorganic acid such as acetate,tartrate, trifluoroacetate, lactate, maleate, fumarate, citrate,methane, sulfonate, sulfate, phosphate, nitrate, or chloride.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the novel dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active material and the particulartherapeutic effect to be achieved and (b) the limitations inherent inthe art of compounding.

Effective amounts contemplated by the present invention will varydepending on the severity of the condition and the health and age of therecipient. In general terms, effective amounts may vary from 0.01 ng/kgbody weight to about 100 mg/kg body weight. Effective amounts includeabout 100 mg to about 600 mg, in particular about 100 mg, about 150 mg,about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg,about 450 mg, about 500 mg, about 550 mg, or about 600 mg.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein are to be understood as modified in all instances by the term“about”.

The recitation of a numerical range using endpoints includes all numberssubsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3,3.80, 4, 5, etc.).

Preferred Embodiment of the Invention

Although the invention has been described with reference to certainembodiments detailed herein, other embodiments can achieve the same orsimilar results. Variations and modifications of the invention will beobvious to those skilled in the art and the invention is intended tocover all such modifications and equivalents.

The present application is based on the surprising finding that BIT225has activity against multiple subtypes and strains of influenza virus.Importantly, BIT225 has been found to have activity against subtypes andstrains that are resistant to Amantadine.

The present invention provides methods and compositions (such aspharmaceutical compositions) for treating or preventing influenza.

The present invention provides materials and methods for preventingand/or treating viral infections. Specifically, the subject inventionprovides materials and methods for preventing influenza infection;treating/ameliorating symptoms associated with influenza infections;and/or preventing/delaying the onset of complications associated withinfluenza infections.

The present invention provides a method for the treatment or preventionof influenza virus infection in a subject, the method comprisingadministering to the subject an effective amount of BIT225, or apharmaceutically acceptable salt thereof.

The present invention also provides a method for inhibiting thereplication of influenza virus in a subject, the method comprisingadministering to the subject an effective amount BIT225, or apharmaceutically acceptable salt thereof.

The present invention further provides a method for reducing theseverity, intensity, or duration of complications or symptoms associatedwith influenza virus infection in a subject, the method comprisingadministering to the subject an effective amount BIT225, or apharmaceutically acceptable salt thereof.

The invention further provides a method of reducing the titre ofinfluenza virus in a subject, the method comprising administering to thesubject an effective amount of BIT225, or a pharmaceutically acceptablesalt thereof.

The present invention is further described by the following non-limitingexamples.

Examples

Production of BIT225

A mixture of 5-bromo-2-naphthoic acid (2.12 g, 8.44 mmol),1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(1.84 g, 8.86 mmol), and tetrakis(triphenylphosphine)palladium(0) (502mg, 0.435 mmol) in a 250 mL round bottomed flask was evacuated andpurged with nitrogen (in three cycles). Acetonitrile (40 mL) and 2Maqueous sodium carbonate (10 mL) were added to the mixture via syringe,and the mixture was heated under reflux under nitrogen for 22 hours. Thereaction mixture was allowed to cool before the addition of 1M aqueoushydrochloric acid (30 mL) and it was then extracted with ethyl acetate(3×50 mL). The combined organic layers were dried (MgSO₄), filtered, andconcentrated in vacuo to provide a crude product (2.98 g after airdrying). This crude material was dissolved in hot ethanol (150 mL) andfiltered while hot to remove a yellow impurity (120 mg). The filtratewas concentrated in vacuo and the residue was recrystallised fromdichloromethane (30 mL) to provide5-(1-methyl-1H-pyrazol-4-yl)-2-naphthoic acid as a white solid (724 mg,34%). A second crop of 5-(1-methyl-1H-pyrazol-4-yl)-2-naphthoic acid(527 mg, 25%) was obtained from the concentrated mother liquors byrecrystallisation from dichloromethane (20 mL).

Oxalyl chloride (1.1 mL, 13 mmol) was added to the solution of5-(1-methyl-1H-pyrazol-4-yl)-2-naphthoic acid (1.19 g, 4.71 mmol) inanhydrous dichloromethane (200 mL (which was added in portions duringthe reaction to effect dissolution)) containing dimethylformamide (2drops) under nitrogen and the mixture was stirred at room temperaturefor 4.25 hours. The reaction mixture was then heated for 1 hour at 40°C., before being concentrated under reduced pressure. The resultingcrude acid chloride was suspended in anhydrous tetrahydrofuran (50 mL)and this mixture was added dropwise to a solution of guanidinehydrochloride (2.09 g, 21.9 mmol) in 2M aqueous sodium hydroxide (15 mL,30 mmol) and the reaction mixture was then stirred for 30 minutes. Theorganic phase was separated, and the aqueous phase was extracted withchloroform (3×30 mL) followed by ethyl acetate (3×30 mL). The combinedorganic extracts were washed sequentially with 1M aqueous sodiumhydroxide (60 mL) and water (40 mL), then dried (Na₂SO₄) andconcentrated in vacuo to give a glassy solid (1.45 g after drying underhigh vacuum). This solid was dissolved in dichloromethane which was thenallowed to evaporate slowly to give BIT225 as a yellow solid (1.15 g,83%).

Virus Strains

The activity of BIT225 was measured against the following viral strains:

-   -   Influenza A/New Caledonia/20/99 (H1N1). Recent clinical isolate        used in latest vaccine (CDC);    -   Influenza A/Panama/2007/99 (H3N2). Recent clinical isolate used        in latest vaccine (CDC);    -   Influenza B/Hong Kong/330/02. Recent clinical isolate used in        latest vaccine (CDC);    -   Influenza A/NWS/33 (H1N1). A well-recognized laboratory strain        (KW. Cochran, Univ. Michigan).

All viral strains were tested in the presence of trypsin, although incertain studies the A/NWS/33 virus was used without trypsin.

Assay of Antiviral Activity:

Rapid Screening Assay

The standard cytopathic effect (CPE) assay uses an 18 hour monolayer(80-100% confluent) of the appropriate cells, medium is removed and eachof the concentrations of test compound or placebo added, followed within15 minutes by virus or virus diluent. Two wells are used for eachconcentration of compound for both antiviral and cytotoxicity testing.The plate is sealed and incubated for the standard time period requiredto induce near-maximal viral CPE. The plate is then stained with neutralred by the method described below and the percentage of uptakeindicating viable cells read on a microplate autoreader at dualwavelengths of 405 and 540 nm, with the difference taken to eliminatebackground. An approximated virus-inhibitory concentration, 50% endpoint(EC₅₀) and cell-inhibitory concentration, 50% endpoint (CC₅₀) aredetermined from which a general selectivity index is calculated:SI=(CC₅₀)/(EC₅₀). An SI of 3 or greater triggers confirmatory testing.

Inhibition of Viral Cytopathic Effect (CPE)

This assay, run in 96 well flat-bottomed microplates, is used for theinitial antiviral evaluation of all new test compounds. In this CPEinhibition test, four log₁₀ dilutions of each test compound (e.g. 1000,100, 10, 1 μg/mL) are added to 3 wells containing the cell monolayer andwithin 5 minutes, the virus is added, the plate sealed, incubated at 37°C. CPE are read microscopically when untreated infected controls developa 3 to 4+ CPE (approximately 72 to 120 hours). A known positive controldrug is evaluated in parallel with test drugs in each assay run.Follow-up testing with compounds found active in initial screening testsare run in the same manner except 8 one-half log₁₀ dilutions of eachcompound are used in 4 wells containing the cell monolayer per dilution.

The data is expressed as 50% effective concentrations (EC₅₀).

Increase in Neutral Red Dye Uptake

This assay is run to validate the CPE inhibition observed in the initialtest, and utilizes the same 96-well micro plates after the CPE has beenevaluated. Neutral red is added to the medium; cells not damaged byvirus take up a greater amount of dye, which is read on a computerizedmicro plate autoreader. The method as described by McManus (McManus, AppEnviron Microbiol, 1976. 31(1): 35-38) is used. An EC₅₀ is determinedfrom the dye uptake.

BIT225 has antiviral activity against both amantadine sensitive andresistant viruses. Its broad-spectrum activity and in particular itsactivity against influenza B make it an important antiviral compound.The antiviral data against influenza A and B for BIT225 in Madin Darbycanine kidney (MDCK) cells is summarized in Table 1. Commerciallyavailable antiviral agents generally do not have antiviral activityagainst influenza B.

TABLE 1 Amantadine EC₅₀ CC₅₀ Com- Virus Sensitivity Assay (μM) (μM) SIments Influenza A Sensitive Neutral Red 22 22 1 Active (H1N1) Visual 432 8 Influenza A Sensitive Neutral Red 2.2 45 20 Active (H3N2) Visual3.2 32 10 Influenza A Resistant Neutral Red 3.2 15 4.5 Active (H5N1)Visual 10 32 3.2 Influenza B Resistant Neutral Red 3.6 17 4.7 ActiveVisual 2.8 32 11Dose Response Plaque Reduction Assay

The anti-influenza A virus activity of BIT225 was confirmed by an invitro dose response plaque reduction assay in Madin Darby canine kidney(MDCK) cells. The laboratory strain PR8 was used in these experimentsand an amantadine resistant variant virus was made by mutating the M2gene to encode sequence threonine at position 30 instead of alanine. The[A30T] mutant virus is known as PR8 4C.

The plaque assays were performed as described by Hayden et al. (Haydenet al., Antimicrob Agents Chemother, 1980. 17(5): 865-870) utilizing1×10³ pfu of the PR8 recombinant viruses in 150 μL of media to infectmonolayers of MDCK cells.

This dilution of virus gives ˜100 plaques in untreated control wells.BIT225 was dissolved in DMSO and added to media at the indicated finalconcentrations. Toxicity controls indicated no toxic effects of BIT225up to 100 μM in MDCK cells.

FIG. 1 illustrates that BIT225 was able to reduce viral replication ofboth amantadine sensitive and resistant PR8 viruses. The EC₅₀ for BIT225was approximately 10 μM against both the wild-type and A30T mutantviruses, with respective EC90 values of 12 μM and 18 μM. This is instark contrast to the large shift in drug sensitivity to amantadinecaused by the A30T mutation in M2.

Chimeric Virus Studies

Experiments were conducted to examine the effect of BIT225 onreplication in a T cell line (C8166) of the chimeric viruses SHIV-1_(M2)and SHIV-1_(SCVpu), which express the ion channel sequences of influenzaA M2 or HIV-1 Vpu, respectively. Treatment with 10 μM BIT225 reducedvirus released into the culture supernatant as measured by viral p27protein (FIG. 2). Controls treated with carrier solvent alone (DMSO) areshown for comparison.

However, treatment with 10 μM BIT225 of the mutant SHIV-1_(PND2) viruswith a non-functional Vpu ion channel does not significantly reducevirus replication (FIG. 3).

Taken together, these results indicate that inhibition of the Vpu or M2ion channels is a possible mechanism of action of BIT225 for influenza.

The invention claimed is:
 1. A method for inhibition of an influenzavirus infection by the influenza virus in a subject, the methodcomprising administering to the subject an effective amount ofN-carbamimidoyl-5-(1-methyl-iH-pyrazol-4-yl)-2-naphthamide, or apharmaceutically acceptable salt thereof, wherein the administeringinhibits replication of the influenza virus in the subject.
 2. Themethod of claim 1, wherein severity, intensity, or duration ofcomplications or symptoms associated with the infection by the influenzavirus are reduced in the subject.
 3. The method of claim 1, whereintiter of the influenza virus is reduced.
 4. The method of claim 1,wherein the influenza virus is influenza A virus, influenza B virus orinfluenza C virus.
 5. The method of claim 1, wherein the influenza virusis influenza A virus.
 6. The method of claim 5 wherein the influenza Avirus is H1N1, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H5N3, H5N6, H5N8,H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, or H10N7 subtype.
 7. Themethod of claim 5, wherein the influenza A virus is H1N1, H3N2 or H5N1subtype.
 8. The method of claim 1, wherein the influenza virus isinfluenza B virus.
 9. The method of claim 1, comprising administeringthe N-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or thepharmaceutically acceptable salt thereof, by an oral, nasal,intravenous, intraperitoneal, inhalation or topical route to thesubject.
 10. The method of claim 1, comprising orally administering theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or thepharmaceutically acceptable salt thereof, to the subject.
 11. The methodof claim 1, comprising administering a dosage of about 100 mg to about600 mg of theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or thepharmaceutically acceptable salt thereof, to the subject.
 12. The methodof claim 1, comprising administering theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or thepharmaceutically acceptable salt thereof, orally, once daily at a dosageof about 100 mg to about 200 mg, to the subject.
 13. The method of claim1, comprising administeringthe-N-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or thepharmaceutically acceptable salt thereof, orally, twice daily at adosage of about 100 mg to about 200 mg, to the subject.
 14. The methodof claim 1, comprising administering theN-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide, or thepharmaceutically acceptable salt thereof, in combination with one ormore additional antiviral agents, to the subject.
 15. The method ofclaim 14, wherein the one or more additional antiviral agents compriseszanamivir, oseltamivir and/or peramivir.